Mutation-induced perturbation of the cytochrome c alkaline transition

Abstract

The possible influence of residue Phe-82 in the cytochrome c alkaline isomerization has been evaluated by spectrophotometric pH titrations of a family of mutant yeast iso-1-cytochromes c in which the identity of the residue at this position has been varied. The pKa for the exchange of the Met-80 heme iron ligand was determined from pH titrations in which the S----Fe charge-transfer band (695 nm) was monitored and was found to be 8.5 for the wild type, 7.7 for Ser-82, 7.7 for Gly-82, 7.2 for Leu-82, and 7.2 for Ile-82. pH-jump experiments [Davis et al. (1974) J. Biol. Chem. 249, 2624] established that substitutions at position 82 affect the alkaline isomerization by lowering the pKa of the titrating group by as much as 1.4 pK units; for the Ser-82 and Gly-82 variants, there is also a small effect on the Keq for the ligand exchange equilibrium. On the basis of these findings, we conclude that one critical role for Phe-82 in the wild-type protein is stabilization of the native heme binding environment.